The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.
The development of companion diagnostics has the potential to improve patient outcomes, and eliminate the need for insurers to pay for expensive yet ineffective therapies. As the number of genes implicated in cancer continues to grow, it is becoming evident that a careful characterization of the genetic alterations that define an individual patient's tumors will often be useful in determining optimal therapeutic strategies. For example, Foundation Medicine® offers a large-scale solid tumor screening panel that interrogates the entire coding sequence of over 300 cancer-related genes and 28 gene rearrangements using DNA or RNA bait libraries (e.g., FoundationOne®). Such assays require a tumor DNA input of at least 50 ng. However, the amount of DNA available for such comprehensive studies is often limited and of poor quality because the tumor DNA is isolated from formalin fixed paraffin-embedded (FFPE) tissues. The FFPE process frequently degrades DNA into small fragments and has the potential to damage the DNA base pairs themselves.
TruSight™ Tumor (Illumina, Inc.) is an example of an existing PCR-based NGS tumor screening panel that interrogates a narrower set of cancer-related genes (174 amplicons within 26 genes) and requires a minimum DNA input of 30 ng. However, this method requires an evaluation of the quality of genomic DNA extracted from the FFPE tumor sample via quantitative PCR prior to generating an amplicon-based library because neither tissue area nor DNA yield are adequate predictors of library performance. See TruSight™ Tumor Data Sheet (Illumina, Inc.); Generating Sequencing Libraries from FFPE Samples, White Paper (Illumina, Inc.).
Detecting actionable genetic alterations in FFPE tissues is further complicated by the fact that cells within a tumor sample can exhibit a high degree of molecular variation between tumors (inter-tumor heterogeneity) and within the individual tumor itself (intra-tumor heterogeneity). Tumor heterogeneity has been observed in leukemias, melanomas, breast, prostate, colon, lung, and gynecological carcinomas. Accordingly, the small fraction of cells in a biopsy may not be representative of the entire tumor mass, which could lead to false negative calls for a given genetic alteration.
Intra-tumor heterogeneity may also explain, at least in part, why some patients who initially respond well to a cancer drug eventually relapse, often with new tumors that no longer respond to the therapy. The higher the diversity of cells within a tumor, the greater the risk that an occasional cell might be able to adapt to the type of stress a drug imposes. Acquired resistance to cancer drugs may develop through a variety of mechanisms (Chong C. & Janne P., Nat. Med. 19: 1389-1400 (2013); Katayama et al., Sci. Transl. Med. 4(120): 120ra17 (2012)). For example, resistant cells can develop a compensatory signaling pathway, or “bypass track,” that reestablishes activation of key downstream proliferation and survival signals despite inhibition of the original oncogene (Niederst & Engelman, Sci. Signal. 6: re6 (2013)). Thus, the heterogeneity of cancer cells introduces significant challenges in designing effective treatment strategies, especially when a specific gene mutation is not detected in the biopsy.
Thus, there is a substantial need for more robust and sensitive methods that effectively detect the presence of genetic alterations in highly heterogeneous tumors samples, particularly in FFPE tissues. Such methods would aid in predicting the responsiveness of individual patients to a particular drug regimen and the identification of optimal therapeutic strategies at the outset.